Plant Biotechnology Journal

(2006)

4

, pp. 251–261 doi: 10.1111/j.1467-7652.2005.00178.x

© 2006 Blackwell Publishing Ltd

251

tBlackwell Publishing, Ltd.Oxford, UKPBIPlant Biotechnology Journal1467-7644© 2005 Blackwell Publishing Ltd? 20052?Original Article

Transformation of barley pollen cultures

Jochen Kumlehn

et al.

Genetic transformation of barley (

Hordeum vulgare

L.) via infection of androgenetic pollen cultures with

Agrobacterium tumefaciens

Jochen Kumlehn

1,2,

*, Liliya Serazetdinova

2,

†, Goetz Hensel

1

, Dirk Becker

2

and Horst Loerz

2

1

Institute of Plant Genetics and Crop Plant Research Gatersleben, Plant Reproductive Biology, Corrensstr. 3, 06466 Gatersleben, Germany

2

University of Hamburg, Biocentre Klein Flottbek, Developmental Biology and Biotechnology, Ohnhorststr. 18, 22609 Hamburg, Germany

Summary

A novel genetic transformation method for barley (

Hordeum vulgare

L.), based on infection

of androgenetic pollen cultures with

Agrobacterium tumefaciens

, is presented. Winter-type

barley cv. ‘Igri’ was amenable to stable integration of transgenes mediated by

A.

tumefaciens

strain LBA4404 harbouring a vector system that confers hypervirulence, or by

the non-hypervirulent strain GV3101 with a standard binary vector. The efficacy of gene

transfer was substantially influenced by pollen pre-culture time, choice of

Agrobacterium

strain and vector system,

Agrobacterium

population density, medium pH and the

concentrations of acetosyringone, CaCl

2

and glutamine. After co-culture, rapid removal of

viable agrobacteria was crucial for subsequent development of the pollen culture. To this

end, the growth of agrobacteria was suppressed by the concerted effects of appropriate

antibiotics, low pH, reduced level of glutamine and high concentrations of CaCl

2

and

acetosyringone. Following infection with LBA4404 and GV3101, about 31% and 69%,

respectively, of the primary transgenic (T

0

) plants carried a single copy of the sequence

integrated. The use of hypervirulent

A. tumefaciens

and hygromycin resistance as a

selectable marker resulted in 3.7 T

0

plants per donor spike. About 60% of the primary

transgenic plants set seed, indicating spontaneous genome doubling. An analysis of 20 T

1

populations revealed that four progenies did not segregate for reporter gene expression.

This indicates that the approach pursued enables the generation of instantly homozygous

primary transgenic plants. The method established will be a valuable tool in functional

genomics as well as for the biotechnological improvement of barley.

Received 12 May 2005;

revised 21 October 2005;

accepted 27 October 2005.

*

Correspondence

(fax +49394825692;

e-mail kumlehn@ipk-gatersleben.de)

†

Present address

: Sainsbury Laboratory,

John Innes Centre, Colney Lane,

Norwich NR4 7UH, Norfolk, UK

Keywords:

Agrobacterium

,

androgenesis, barley, genetic

transformation,

Hordeum vulgare

,

pollen embryogenesis.

Introduction

Reliable and efficient techniques to genetically transform

important experimental model plant species, as well as crops,

are required for comprehensive functional gene analyses and

molecular breeding. As an important food and feed crop grown

worldwide, barley has been used as a model species in cereal

genetic research for decades. The barley genetic resources

generated to date (e.g. Close

et al

., 2004; Zhang

et al

., 2004)

are now being intensively employed in diverse research

approaches and, for many sequences, a detailed functional

characterization via stable genetic transformation is desirable.

Stable transformation of barley has been performed by

direct DNA transfer to immature embryos (Wan and Lemaux,

1994), androgenetic pollen (Jaehne

et al

., 1994), shoot mer-

istematic cultures (Zhang

et al

., 1999), protoplasts (Funatsuki

et al

., 1995) or isolated zygotes (Holm

et al

., 2000). Tingay

et al

. (1997) were the first to establish a protocol for

Agro-

bacterium

-mediated transformation of barley. Later, basically

the same method, which is based on the infection of imma-

ture embryos with agrobacteria, was also employed in other

laboratories (Horvath

et al

., 2000; Patel

et al

., 2000; Trifonova

et al

., 2001; Wang

et al

., 2001; Fang

et al

., 2002; Stahl

et al

.,

2002). In more recently published reports, transformation

252

Jochen Kumlehn

et al.

© Blackwell Publishing Ltd,

Plant Biotechnology Journal

(2006),

4

, 251–261

efficiencies higher than 10%, based on the number of in-

fected embryos, were achieved (Matthews

et al

., 2001; Hensel

and Kumlehn, 2004; Murray

et al

., 2004).

Agrobacterium

-

mediated transformation of cereals is still confined to a few

selected genotypes. For example, in barley, only a few spring-

type lines have been found to be useful to some extent (Tingay

et al

., 1997; Wang

et al

., 2001; Murray

et al

., 2004), whereas,

so far, there is no example of genetic transformation of a

barley winter-type line. In particular, the spring-type cv. ‘Golden

Promise’ is extraordinarily amenable to

Agrobacterium

-

mediated transformation of immature embryos.

In comparison with direct gene transfer methods,

Agrobacterium

-mediated transformation is basically governed

by a naturally evolved mechanism (Tinland, 1996; Zupan and

Zambryski, 1997). It predominantly results in the integration

of single or low copy numbers of transferred DNA per

transformed cell, which supports expression stability in

subsequent generations. Barakat

et al

. (2000) revealed

a predisposition of agrobacteria to integrate T-DNAs into

transcriptionally active regions of the rice genome. Further

advantages of

Agrobacterium

-mediated transformation

include the high potential for independent integration of

co-transformed DNA fragments, the transfer of large DNA

sequences (Hamilton, 1997) and the exclusion of backbone

sequences from the DNA introduced into the plant genome.

Immature barley pollen at the vacuolated microspore or

early bicellular stage can deviate from the normal process of

pollen formation to undergo androgenesis (Sunderland

et al

.,

1978). The most interesting aspect of genetic transformation

of haploid target cells, such as microspores, is that transgene

integration followed by genome doubling opens up the unique

opportunity to immediately generate plants homozygous for

the gene transferred. In addition to their haploid nature, the

incomparable potential for mass regeneration makes immature

pollen cultures an attractive target for

Agrobacterium

-based

transformation. Sangwan

et al

. (1993) showed the generation

of transgenic

Datura innoxia

and

Nicotiana tabacum

plants

following

Agrobacterium

infection of cotyledonary-stage

androgenetic embryos; however, no transformation was

obtained using freshly isolated immature pollen or andro-

genetic pro-embryos. Successful

A. tumefaciens

-mediated

transformation of barley pollen-derived cell suspension

cultures, obtained by Wu

et al

. (1998), did not result in the

regeneration of transgenic plants. Reports on the generation

of transgenic

Brassica napus

plants following co-culture of

androgenetic pollen with

A. tumefaciens

, published by Pechan

(1989) and disclosed by Dormann

et al

. (2001), lacked com-

pelling evidence for the integration of transgenes in the plant

genome, their inheritance in following generations and the

instant homozygosity of the genes transferred. In the present

study, we have identified and optimized parameters influenc-

ing gene transfer and target cell survival to eventually estab-

lish an efficient and reproducible method of generating

transgenic barley following infection of androgenetic pollen

cultures with agrobacteria. We show that the transgenes are

stably integrated into the barley genome, that these genes

are expressed and inherited in the following generations, and

that some of the primary transgenic plants obtained are

homozygous for the gene transferred.

Results and discussion

Co-culture of androgenetic barley pollen with

A. tumefaciens

Preliminary experiments were carried out with varying pollen

pre-culture times prior to co-culture with the

A. tumefaciens

strain LBA4404 carrying the co-integrative vector pSB1:UG35PAT.

Infection of pollen within the first 5 days after isolation

resulted in no, or extremely rare, transformation events. The

optimal temporal window of efficient infection with agro-

bacteria was determined to be after 6–11 days of pre-culture

(Figure 1A,B).

At least three classes of environmental stimuli act syner-

gistically to induce the transformation machinery of

A. tume-

faciens

, namely monocyclic aromatic hydrocarbons, such as

acetosyringone, monosaccharides, such as glucose, and

acidic pH (Winans, 1991). Acetosyringone plays a dual role in

Agrobacterium

-mediated transformation. First, it serves as a

chemotacticum for the spatial orientation of agrobacteria to

wounded sites of plant tissue (Ashby

et al

., 1987). Second, it

elicits the agrobacterial virulence machinery (Stachel

et al

.,

1985). In addition, we have shown that acetosyringone not

only enhances the transformation activity of

A. tumefaciens

(Figure 2A), but can also substantially limit its multiplication.

Figure 1C displays the overgrowth of the pollen culture in the

absence of acetosyringone, whereas the bacterial growth in

direct contact with the Petri dish, as well as the aggregation

of the androgenetic pollen grains, markedly decreased with

increasing concentration of acetosyringone (Figure 1D–F).

However, concentrations beyond 0.5 m

M

did not further

improve the transformation efficiency (Figure 1E,F).

The moderately acidic pH in the vicinity of wounded plant sites

has been found to activate

A. tumefaciens Vir

genes (Li

et al

.,

2002). Our preliminary attempts to effectively stabilize the

medium pH as a prerequisite for subsequent optimization

revealed that a combination of 10 m

M

2-morpholinoethan-

esulphonic acid (MES) and 50 m

M

potassium hydrogen

Transformation of barley pollen cultures

253

© Blackwell Publishing Ltd,

Plant Biotechnology Journal

(2006),

4

, 251–261

Figure 1 Agrobacterium-mediated transformation of barley pollen cultures. (A) Androgenetic barley pollen suitable for infection with agrobacteria after 7 days of culture. (B) Barley pollen culture 2 weeks after co-culture with Agrobacterium tumefaciens. Fluorescence of green fluorescent protein (GFP) in developing microcalluses (green) can be easily distinguished from self-fluorescing pollen and tissue (yellowish). (C–F) Histochemical assessment of β-glucuronidase (GUS) expression 7 days after co-culture in the presence of 0 (C), 0.2 (D), 0.5 (E) and 0.7 mM (F) acetosyringone. Note that 0.5 mM acetosyringone results in the highest transformation efficiency and that pollen aggregation, which is dependent on bacterial growth, decreases with increasing acetosyringone concentration. (G) Plant regeneration from barley pollen cultures after co-culture with LBA4404(pSB1/pYF133) and 4 weeks of selection with hygromycin in liquid medium. (H) Southern blot showing hybridization of a (32P)-dCTP-labelled GUSi probe with SpeI-digested genomic DNA of 15 randomly chosen T0 plants obtained following co-culture of androgenetic pollen with GV3101(pUGAB7). C–, negative control with genomic DNA of a wild-type cv. ‘Igri’ plant, C+, positive control with binary plasmid DNA of pUGAB7. (I) Roots of a wild-type plant (below) and of a plant resulting from pollen transformation with LBA4404(pSB1/pYF133) (above) shown by an inverted microscope under white light. (J) The same objects as shown in (I) using ultraviolet (UV) light and an appropriate filter set. Note that GFP-expressing plants can be unambiguously discriminated from non-transgenic plants by this method. (K) A T2 plant-derived pollen population segregating 1 : 1 for GFP expression shown under UV light and an appropriate filter set.

254

Jochen Kumlehn

et al.

© Blackwell Publishing Ltd,

Plant Biotechnology Journal

(2006),

4

, 251–261

phosphate was effective in retaining the pH within a range of

±

0.1 pH units during 2 days of co-culture, provided that the

initial density of agrobacteria was not higher than 10

7

colony-

forming units (cfu)/mL. A subsequent experiment showed

that a stabilized pH of about 5.9 represented an optimum for

the transformation of barley pollen cultures by

Agrobacterium

strain LBA4404(pSB1:UG35PAT) (Figure 2B). In the following

experiments, pH 5.9 also supported efficient transformation

using LBA4404(pSB1/pYF133) or GV3101(pUGAB7). By con-

trast, Turk

et al

. (1991) reported that different types of

Agro-

bacterium

strain showed different pH sensitivity patterns.

In addition, Godwin

et al

. (1991) found that the virulence of

A. tumefaciens

was affected by medium pH; however, there

was no general effect across plant species. Our experiment

on the optimization of the medium pH revealed that both

gene transfer and agrobacterial growth were strongly influ-

enced in a pH-dependent manner. The intensity of bacterial

growth could be easily observed, as in the previous experi-

ment on the optimization of the acetosyringone concentra-

tion (Figure 1C–F). In contrast with conventional plant cell

culture media, agrobacterial growth was considerably less

intense on effective pH stabilization in the range pH 5–6 using

the above-mentioned buffers, resulting not only in increased

transformation efficiency, but also in substantially improved

survival of androgenetic microcalluses. Montoro

et al

. (2000)

reported that CaCl

2

markedly influenced the

Agrobacterium

-

mediated transformation of

Hevea brasiliensis

. By contrast

with their experiments, we obtained no transformation

events on co-culture without CaCl

2

, and found 3 m

M

CaCl

2

to be optimal for barley pollen transformation (Figure 2C).

Moreover, we ascertained that the CaCl

2

concentration was

negatively correlated with agrobacterial growth, whereas a

concomitant decrease in transformation efficiency accom-

panied CaCl

2

concentrations higher than 3 m

M

.

In another experimental approach, we investigated the

impact of glutamine on

Agrobacterium

-mediated transfor-

mation. Glutamine is essential in androgenetic pollen cul-

tures of numerous species, including barley (Olsen, 1987),

and agrobacteria can use glutamine as both a nitrogen and

carbon source. Accordingly, we found that glutamine mark-

edly supported the growth of

A. tumefaciens

in co-culture.

Figure 2

Effect of (A) acetosyringone, (B) pH, (C) CaCl

2

and (D) L-glutamine in the co-culture medium on the transformation of androgenetic barley pollen with Agrobacterium tumefaciens LBA4404(pSB1:UG35PAT). Independent transformation events were scored 1 week after co-culture by histochemical assessment of β-glucuronidase (GUS) expression in six replications per treatment. The columns represent median values in experiments in which the normality test had failed (A, C) or mean values in cases in which both the normality

and equal variance tests (P < 0.05) were passed (B, D). Significant differences between treatments (P < 0.05), as indicated by the different colours of the respective columns, were calculated using Tukey’s all pairwise multiple comparison procedure following either Kruskal–Wallis analysis of variance (ANOVA) on ranks (A, C) or conventional ANOVA (B, D).

Transformation of barley pollen cultures 255

© Blackwell Publishing Ltd, Plant Biotechnology Journal (2006), 4, 251–261

Furthermore, our study demonstrated that glutamine did not

enhance the transformation activity of agrobacteria and, sur-

prisingly, it appeared not to be required during the 2 days of

co-culture for androgenetic development to proceed (Figure 2D).

Effect of Agrobacterium strain and vector system

Transgenic barley plants were produced using three different

Agrobacterium strain–vector combinations. The generation

of a superbinary vector, as suggested by Komari et al. (1996),

was comparatively intricate and we were unable to maintain

the resultant strain LBA4404(pSB1:UG35PAT) for more than

a few months. As a consequence, we pursued an alternative

approach based on the introduction of the non-integrative

binary vector pYF133 (Fang et al., 2002) into the hypervirulent

strain LBA4404(pSB1). The resultant LBA4404(pSB1/pYF133)

was the most efficient with regard to the generation of trans-

genic plants (Table 1). The substantially lower transformation

efficiency obtained with LBA4404(pSB1:UG35PAT) can prob-

ably be attributed to the PAT gene used as a selectable

marker. However, effects associated with the different vector

construction cannot be ruled out. In addition to hypervirulent

agrobacteria, we tested the non-hypervirulent A. tumefa-

ciens strain GV3101 containing the standard binary vector

pUGAB7. As a result, transformation of barley pollen cultures

was obtained with an efficiency comparable with that of

LBA4404(pSB1:UG35PAT) (Table 1).

Removal of agrobacteria and selective development of

transgenic tissue and plants

Based on the results from the co-culture experiments, the ASt

medium composition (see Table 3) was optimized in prelim-

inary small-scale experiments in order to effectively suppress

agrobacterial growth immediately after transformation, as

well as to ensure ongoing androgenetic development. In

particular, the pH was adjusted to 5.0 and stabilized using

10 mM MES together with 25 mM potassium hydrogen phos-

phate, the CaCl2 concentration was increased to 10 mM, ace-

tosyringone was added to obtain a concentration of 0.5 mM,

and glutamine was added to obtain a concentration of 1 mM.

These treatments markedly contributed to the control of

agrobacteria, whereas the androgenetic development was

apparently not adversely affected (data not shown). Further-

more, a combination of the antibiotics amoxicillin (100 mg/L)

and cefotaxime (125 mg/L) was successfully used to remove

viable agrobacteria. After 2 weeks of culture in ASt medium,

we checked the presence of viable agrobacteria by transfer-

ring some pollen-derived calluses on to solid Luria–Bertani

(LB) medium without the addition of antibiotics. Not a single

case of outgrowing agrobacteria was detected, indicating their

effective removal under the conditions used after co-culture.

Hygromycin (50 µM) or Bialaphos (75 µM), added immediately

after co-culture, was useful to prevent substantial formation

of non-transgenic tissue and plants. One week after co-culture,

the transgenic microcalluses tolerated even further increased

concentrations of selective agent. Accordingly, S medium

(see Table 3) was supplemented with 100 µM of either hygro-

mycin or Bialaphos.

A considerable delay in the generation of plantlets from

transformed pollen cultures was observed when selective

pressure was applied during regeneration in preliminary

experiments. The experiments presented here demonstrate

that plantlet formation is more efficient and the proportion

of non-transgenic ‘escapes’ remains low when the selective

agent is entirely omitted from the regeneration medium

(Figure 1G, Table 1). This observation can be explained by the

strong selective pressure during previous liquid culture.

Integration of transgenes into the barley genome and

expression of recombinant protein

All candidate primary transgenic plants were analysed using

Southern blotting to reveal stable transgene integration within

Table 1 Formation of transgenic plants on Agrobacterium-mediated transformation of androgenetic pollen cultures of barley

Strain/plasmid

Pollen

processed

(Mio)

Resistant

plants

obtained

Southern-

positive T0

plants

T0 plants per

Mio pollen

T0 plants

per spike

Fertile

T0 plants

Fertile T0

plants per

Mio pollen

Fertile T0

plants

per spike

LBA4404(pSB1:UG35PAT) 10 18 18 (100%) 1.8 0.5 13 (72.2%) 1.3 0.3

LBA4404(pSB1/pYF133) 2.25 35 33 (94.3%) 13.2 3.7 20 (60.1%) 8.9 2.2

GV3101(pUGAB7) 22.75 70 67 (95.7%) 2.9 0.7 36 (53.7%) 1.6 0.4

Total 35 123 118 (95.9%) 3.4 0.84 69 (58.5%) 1.97 0.5

Mio, million.

256 Jochen Kumlehn et al.

© Blackwell Publishing Ltd, Plant Biotechnology Journal (2006), 4, 251–261

their genome (Figure 1H). In the case of transformation using

LBA4404(pSB1/pYF133), followed by selection based on

hygromycin, transgene integration was missing in only two of

35 regenerated plants (Table 1). Compared with hygromycin,

the effect of Bialaphos was markedly less efficient in vitro.

Using GV3101(pUGAB7), a total of 174 plantlets was

regenerated, only 70 (40.2%) of which survived spraying

with BASTA solution. Yet, about 96% and 100% of the

surviving plants derived from co-culture using GV3101(pSB1/

UGAB7) or LBA4404(pSB1:UG35PAT), respectively, showed

transgene integration according to Southern hybridization

(Table 1), indicating that spraying with BASTA can be

considered as a fairly reliable method. Southern blotting

analyses further revealed that 16 of 51 (31.4%) and 46 of 67

(68.6%) T0 plants obtained following co-culture with

LBA4404 and GV3101, respectively, carried a single copy of

the transferred T-DNA. Expression of green fluorescent

protein (GFP) was unambiguously shown by fluorescence

microscopy of root tips in 93.9% of Southern-positive T0

plants transformed with LBA4404(pSB1/pYF133) (Figure 1I,J ).

Generative transgene transmission to T1 plants was also

revealed by Southern blotting (not shown). Fluorescence

microscopy further demonstrated one-to-one segregation of

GFP expression in pollen populations of hemizygous single-

copy T2 plants, which provides evidence for sustainable trans-

mission, segregation and expression of the integrated GFP

gene (Figure 1K).

Considering that spontaneous genome doubling appears

to preferentially occur early in androgenetic development,

and that transformation was feasible only after a consider-

able period of pollen pre-culture time, it was surprising that

four of 20 T1 populations did not segregate for marker

gene expression (Table 2), thus indicating that spontaneous

genome doubling must have taken place in some cases

subsequent to the transformation event. Such late spontaneous

genome doubling events have recently been observed by

González-Melendi et al. (2005).

Conclusions

A novel technology has been established for Agrobacterium-

based genetic transformation of barley. Compared with

immature embryo-based transformation, the system pre-

sented here has several advantages. The spikes are harvested

at a markedly earlier developmental stage, i.e. a higher

throughput of donor plants can be obtained with a given

glasshouse capacity, and, as the donor plants can be dis-

carded prior to anthesis, phytopathogens can be controlled

more easily. Moreover, the microspores from 30 spikes can

be isolated and transferred to culture within about 1 h. Given

Table 2 Transgene segregation in 20 T1 populations derived from Agrobacterium-mediated transformation of barley pollen cultures

T0 plants

Reporter gene

integrated

No. of copies

according to

Southern blot

Number of T1

plants analysed

Reporter gene

expression/no

expression

Segregation

observed

(assumed)

χ2

value

Likelihood (P)

according to

χ2 test

1 GUSi 1 20 14 : 6 2.33 : 1 (3 : 1) 0.24 > 0.5

2 GUSi 1 24 18 : 6 3.0 : 1 (3 : 1) 0 > 0.99

3 GUSi 2 43 37 : 6 6.17 : 1 (15 : 1) 1.9 > 0.1

4 GUSi 1 26 17 : 9 1.89 : 1 (3 : 1) 1.06 > 0.3

5 GUSi 2 39 34 : 5 6.8 : 1 (15 : 1) 1.32 > 0.2

6 GUSi 1 22 22 : 0 22 : 0 (22 : 0) 0 > 0.99

7 GUSi 1 51 40 : 9 3.64 : 1 (3 : 1) 0.35 > 0.5

8 GUSi 1 51 34 : 17 2.0 : 1 (3 : 1) 1.59 > 0.2

9 GUSi 1 50 32 : 18 1.78 : 1 (3 : 1) 2.63 > 0.1

10 GUSi 1 53 42 : 11 3.82 : 1 (3 : 1) 0.58 > 0.3

11 GUSi 1 16 16 : 0 16.0 : 0 (16 : 0) 0 > 0.99

12 GFP 1 46 35 : 11 3.18 : 1 (3 : 1) 0.03 > 0.8

13 GFP 2 43 37 : 6 6.17 : 1 (15 : 1) 1.9 > 0.1

14 GFP 2 27 23 : 4 5.75 : 1 (15 : 1) 1.42 > 0.2

15 GFP 1 20 20 : 0 20.0 : 0 (20 : 0) 0 > 0.99

16 GFP 1 18 12 : 6 2.0 : 1 (3 : 1) 0.56 > 0.3

17 GFP 2 21 19 : 2 9.5 : 1 (15 : 1) 0.2 > 0.5

18 GFP 1 24 24 : 0 24.0 : 0 (24 : 0) 0 > 0.99

19 GFP 2 14 12 : 2 6.0 : 1 (15 : 1) 0.66 > 0.3

20 GFP 2 19 16 : 3 5.3 : 1 (15 : 1) 1.19 > 0.2

Transformation of barley pollen cultures 257

© Blackwell Publishing Ltd, Plant Biotechnology Journal (2006), 4, 251–261

10 transgenic plants per 100 embryos and 15 caryopses used

per donor spike in Agrobacterium-mediated transformation

using barley immature embryos, the efficiency of 2.2 fertile

transgenic plants obtained per spike in this study compares

well with that obtained by embryo transformation. The par-

ticular value of the method established in this work is that a

considerable number of plants which are homozygous for

the gene transferred can be found amongst the primary

transgenics. Although segregation analysis is also necessary

in this approach, homozygous plants can be identified one

generation earlier in comparison with conventional transfor-

mation methods.

There are several reasons why further improvements of the

newly developed technology can be expected. The majority

of spring- and winter-type genotypes are amenable to

doubled haploid formation via pollen culture. This appears to

be promising with regard to further transformation experi-

ments of diverse barley breeding lines. The surprisingly high

proportion of haploid primary transformants, together with

the amenability of barley to induced genome doubling

Component (mg/L) KBP CK ASt S K4NBCAx

NH4NO3 80 80 80 80 320

KNO3 2022 2022 2022 2022 3640

KH2PO4 340.2 6465 3658 340 340

K2HPO4 – 871 108.9 – –

CaCl2·2H2O 441 441 1470 441 441

MgSO4·7H2O 246 246 246 246 246

NaFeEDTA 27.5 27.5 27.5 27.5 27.5

MnSO4·4H2O 11.2 11.2 11.2 11.2 11.2

H3BO3 3.1 3.1 3.1 3.1 3.1

ZnSO4·7H2O 7.2 7.2 7.2 7.2 7.2

Na2MoO4·2H2O 0.12 0.12 0.12 0.12 0.12

CuSO4·5H2O 0.025 0.025 0.025 0.025 1.25

CoCl2·6H2O 0.024 0.024 0.024 0.024 0.024

KI 0.17 0.17 0.17 0.17 0.17

Retinol 0.01 0.01 0.01 0.01 –

Thiamine-HCl 1.0 1.0 1.0 1.0 10.0

Nicotinic acid 1.0 1.0 1.0 1.0 1.0

Riboflavin 0.2 0.2 0.2 0.2 –

Ca-pantothenate 1.0 1.0 1.0 1.0 –

Folic acid 0.4 0.4 0.4 0.4 –

Pyridoxine-HCl 1.0 1.0 1.0 1.0 1.0

Cobalamine 0.02 0.02 0.02 0.02 –

Ascorbic acid 2.0 2.0 2.0 2.0 –

Calciferol 0.01 0.01 0.01 0.01 –

Biotin 0.01 0.01 0.01 0.01 –

Cholin chloride 1.0 1.0 1.0 1.0 –

p-Aminobenzoic acid 0.02 0.02 0.02 0.02 –

Myo-inositol 100 100 100 100 –

Glutamine 438 – 146 – –

Maltose-H2O 90000 90000 90000 90000 36000

BAP 0.9 0.45 0.45 0.225 0.225

2,4-D – – 2.21 – –

Acetosyringone – 98.1 98.1 – –

Cefotaxime – – 125 125 125

Amoxicillin – – 100 100 100

Bialaphos* – – 24.8 33 –

Hygromycin* – – 26.4 52.8 –

MES – 1952 1952 1952 –

Phytagel – – – – 4.000

pH 5.8 5.9 5.0 5.5 5.8

BAP, 6-benzylaminnopurine; 2,4-D, 2,4-dichlorophenoxyacetic acid; EDTA. ethylenediaminetetraacetate;

MES, 2-morpholinoethanesulphonic acid.

*Bialaphos or hygromycin was used following transformation with the BAR or HPT gene, respectively.

Table 3 Media used for Agrobacterium-mediated transformation of barley pollen cultures and subsequent regeneration of transgenic plants

258 Jochen Kumlehn et al.

© Blackwell Publishing Ltd, Plant Biotechnology Journal (2006), 4, 251–261

techniques, opens up the opportunity for directed and exclusive

formation of doubled haploid T1 seeds instantly homozygous

for the transgene. This could be obtained by flow-cytometric

selection of haploid primary transgenic plants at an early

stage of development, followed by treatment with colchi-

cine. As haploid plants are sterile, seeds will be produced only

from doubled haploid spikes or flowers, resulting in exclusive

formation of homozyous transgenic offspring. However, this

approach requires experimental testing.

Because androgenesis and Agrobacterium-mediated trans-

formation are feasible for many angiosperms, the method

described here is anticipated to have potential for other plant

species.

The method established here has been shown to be repro-

ducible in both laboratories that contributed to this study. Based

on the protocol presented here, several hundred transgenic

barley plants have been produced within the framework of

current studies on promoter characterization, over-expression

and knock-down of genes of interest. Moreover, the method was

successfully employed in a mutant complementation approach

using the barley EIF4E gene, which was convincingly proven

to be associated with Bymovirus resistance (Stein et al., 2005).

Experimental procedures

Plant material

Barley (Hordeum vulgare L.) plants cv. ‘Igri’ (Saatzucht Acker-

mann, Irlbach, Germany) were germinated in a growth

chamber (14/12 °C day/night, 16 h light cycle), followed by

8 weeks vernalization treatment (2 °C, 9 h light cycle) and

cultivation in a controlled glasshouse (18/14 °C day/night, 16 h

light cycle). Artificial illumination was provided by SON-T-

Agro lamps (Philips, Hamburg, Germany) at about 200 W/m2.

Bacterial strains and vectors

The A. tumefaciens octopine strain LBA4404 and the nopaline

strain GV3101, carrying the disarmed Ti plasmids pAL4404 and

pMP90RK, respectively, were used (Hellens et al., 2000). The

LBA4404 strain employed in this study additionally contains

the acceptor vector pSB1 (kindly provided by Japan Tobacco

Inc., Higashibara, Japan). This plasmid harbours homologous

recombination sites for the introduction of a T-DNA region of

choice (Komari et al., 1996) and confers hypervirulence as a

result of the presence of accessory alleles of VirB, VirC and VirG.

The Escherichia coli β-glucuronidase gene (GUS), containing

the first intron of the Solanum tuberosum LS1 gene (Vancanneyt

et al., 1990), was introduced as a BamHI/SacI fragment

between the maize ubiquitin-1 promoter with first intron

(UBIp) and the A. tumefaciens nopaline synthase terminator

(NOSt) into the vector pUBI.cas (D. Becker, unpublished). The

resultant UBIp::GUSi::NOSt expression cassette obtained by

HindIII/EcoRI digestion, as well as an EcoRI fragment from the

vector p35SPAT (kindly provided by P. Eckes, Bayer Crop

Science, Frankfurt, Germany) harbouring the Streptomyces

viridochromogenes phosphinothricin acetyltransferase gene

(PAT) flanked by the promoter and the terminator of the cau-

liflower mosaic virus (CaMV) 35S gene, were introduced into

the T-DNA region of the shuttle vector pSB11 (Komari et al.,

1996; kindly provided by Japan Tobacco Inc.). The tail-to-tail

orientation of the expression cassettes within the generated

vector pUG35PAT was confirmed by mini-preparations and

restriction digests. Unlike pUC, pSB11 retains mob function

of pBR322 (Sambrook et al., 1989), and can be mobilized

by conjugal helper plasmids, such as pRK2013. The pSB11-

derived vector pUG35PAT was introduced via triparental

mating to LBA4404(pSB1). LBA4404 strains with co-integration

of pUG35PAT and pSB1 via homologous recombination were

selected on LB medium (Sambrook et al., 1989) containing

30 µg/mL spectinomycin and 10 µg/mL tetracycline.

The T-DNA of pYF133 includes a synthetic GFP gene driven

by the maize ubiquitin-1 promoter with first intron and an HPT

selectable marker gene under the control of an enhanced CaMV

35S promoter (Fang et al., 2002). The T-DNA of pUGAB7 (DNA

Cloning Service, Hamburg, Germany) contains the GUSi gene

driven by the maize ubiquitin-1 promoter with first intron and

the Streptomyces hygroscopicus BAR gene downstream of

the CaMV 35S promoter.

Aliquots (1 µg) of plasmid DNA from the binary vectors

pUGAB7 and pYF133 were added to 100 µL of frozen compe-

tent cells of LBA4404(pSB1) and GV3101, respectively. The cells

were thawed by incubation for 5 min at 37 °C and then placed

on ice for 30 min. Subsequently, the cells were spread on LB

plates containing the appropriate antibiotics and incubated for

2 days at 28 °C. The presence of a binary vector in the colonies

obtained was confirmed by isolation of plasmid DNA and

restriction analysis. Agrobacteria were maintained on stock

plates containing Agrobacterium broth (AB) medium (Chilton

et al., 1974) supplemented with appropriate antibiotics. The

plates were incubated for 2 days at 28 °C and then stored for

up to 4 weeks at 4 °C.

Prior to infection of androgenetic pollen, a loopful of agro-

bacteria was cultured overnight in 8 mL of liquid AB medium

supplemented with appropriate antibiotics. Grown bacteria were

spun down at 3220 g for 10 min, the pellet was resuspended

in 5 mL of AB6 medium (liquid AB medium containing 50 mM

potassium hydrogen phosphate and adjusted to pH 6) and

Transformation of barley pollen cultures 259

© Blackwell Publishing Ltd, Plant Biotechnology Journal (2006), 4, 251–261

incubated in a 9-cm Petri dish at 21 °C with shaking at

50 r.p.m. for 1–5 h.

Microspore isolation and culture

Spikes were harvested when the tips of the awns had just

emerged from the sheath of the flag leaf. At this develop-

mental stage, the central two-thirds of the florets mainly con-

tain highly vacuolated microspores immediately before the

first pollen mitosis. After removal of the leaf sheaths under

sterile conditions, the awns were removed with forceps. The

spikes were then incubated on a wet filter paper disc in 9-cm

sealed Petri dishes at 4 °C for 3–5 weeks to stimulate andro-

genetic development. To isolate immature pollen, 10 spikes

were chopped down to about 1-cm segments and trans-

ferred to a Waring blender (Eberbach, Ann Arbor, MI, USA)

pre-cooled in a refrigerator. Tissue homogenization was

carried out with 18 mL of ice-cold 0.4 M mannitol twice for

5 s at ‘low’ speed. The homogenate was poured through

a 100-µm nylon mesh (Wilson, Nottingham, UK) into a plastic

container on ice. The debris retained on the mesh was gently

squashed with forceps, rinsed with 10 mL of mannitol solu-

tion and transferred back into the blender. Homogenization

was repeated using 12 mL of 0.4 M mannitol solution and the

debris was washed again with 10 mL. The pollen suspension

collected in the container was transferred into a 50-mL tube

and centrifuged at 100 g for 10 min at 4 °C. After removal of

the supernatant, the pellet was resuspended in 8 mL of ice-

cold 0.55 M maltose. This volume was distributed into two

15-mL tubes and each aliquot was cautiously overlain with

1.5 mL of 0.4 M mannitol. After density gradient centrifuga-

tion at 4 °C and 100 g in a swing-out rotor for 10 min, the

nearly pure population of highly vacuolated microspores was

drawn up from the interphase and suspended in a final vol-

ume of 20 mL filled up with 0.4 M mannitol in a 50-mL tube.

Prior to final pelleting of the immature pollen at 100 g for

10 min, two 15-µL samples were taken to assess the total

number of pollen using a haemocytometer (Paul Marienfeld,

Lauda-Königshofen, Germany). The pellet was diluted in an

appropriate volume of KBP medium (Table 3) to obtain a final

population density of 2.5 × 105 pollen per millilitre. One-

millilitre aliquots were cultured in 35-mm sealed Petri dishes

at 26 °C in the dark until further processing.

Agrobacterium-mediated transformation of pollen

cultures

Six to eleven days after pollen isolation, KBP medium was

removed from the pollen cultures using a disposable pipette

and replaced by 1 mL of CK medium (Table 3). The first

experiments on the optimization of the co-culture conditions

were carried out using a preliminary composition of CK

medium including 0.3 mM acetosyringone, pH 5.8 and 3 mM

glutamine. According to the experimental results, a step-by-

step optimization of the co-culture medium was conducted

to eventually yield the composition of CK medium as given

in Table 3. The population density of the Agrobacterium

pre-culture was determined using a spectrophotometer,

and an appropriate volume was added to a 1-mL culture of

androgenetic pollen to obtain a bacterial density of 108 cfu/

mL, if not stated otherwise. The dishes were incubated at

21 °C with shaking at 65 r.p.m. After 48 h of co-culture, the

medium was removed by means of a disposable pipette,

the pollen was washed in 0.4 mL of ASt medium and sub-

sequently cultured in 1.1 mL of ASt medium supplemented

with the appropriate selective agents (Table 3). After an incu-

bation period of 7 days at 26 °C with shaking at 65 r.p.m.,

ASt medium was replaced either by staining buffer according

to Jefferson (1987) for the assessment of GUS expression, or

by 1.5 mL of S medium (Table 3) for further development.

Sealed culture dishes were kept at 26 °C with further shaking

at 65 r.p.m. S medium was renewed every 7 days. Four

weeks after co-culture, the grown calluses were plated on to

K4NBCAx medium (Table 3) for regeneration. After 1 week

of incubation at 26 °C in the dark, the plates were transferred

into the light. After 4 weeks on K4NBCAx, the developing

structures were transferred to plastic containers with the

same medium and cultured for another 4 weeks. If necessary,

structures were cultured for an additional 4 weeks on fresh

medium prior to the plantlets being potted in soil.

The experiments on the optimization of the co-culture con-

ditions were based on six dishes per treatment. For each A.

tumefaciens strain–binary vector combination used for stable

transformation, at least four independent experiments, each

comprising several 1-mL co-cultures, were performed. GUS

activity was assayed histochemically as described by Jefferson

(1987). GFP expression in callus, root tips and pollen was

visualized by an Axiovert 200M inverted microscope equipped

with epifluorescence (Carl Zeiss, Oberkochen, Germany).

Regenerated plants putatively carrying the selectable marker

genes PAT or BAR were sprayed twice with a 150 mg/L solution

of the herbicide BASTA (AgrEvo, Düsseldorf, Germany) with

an interval of about 7 days.

DNA isolation and Southern blotting analysis

Total genomic DNA was isolated from young leaves as

described by Palotta et al. (2000). Genomic DNA (15–20 µg)

260 Jochen Kumlehn et al.

© Blackwell Publishing Ltd, Plant Biotechnology Journal (2006), 4, 251–261

was digested at 37 °C for 5–6 h with enzymes that had unique

restriction sites in the corresponding T-DNA region. Genomic

DNA of non-transformed cv. ‘Igri’ plants digested with the

same enzymes served as negative control, and 80 pg of the

corresponding restricted plasmid DNA served as positive con-

trol. The digested DNA was size-fractionated by gel electro-

phoresis using 0.8% (w/v) agarose and capillary blotted as

essentially described by Sambrook et al. (1989). For radioactive

blots, DNA was transferred on to Hybond NX nylon membranes

(Amersham, Braunschweig, Germany) which were then

hybridized to (32P)-dCTP-labelled probes synthesized from

polymerase chain reaction (PCR) fragments using a Prime-It II

Random Primer Labelling Kit (Stratagene, Heidelberg, Germany).

Non-incorporated nucleotides were removed with HR S300

microspin columns (Amersham Pharmacia Biotech, Freiburg,

Germany). A GFP probe (0.54 kb) was PCR amplified using the

forward primer 5′-GCGACGTAAACGGCCACAAGTTCA-3′and the reverse primer 5′-TAGTGGTTGTCGGGCAGCAGC-3′.A GUS-intron probe (0.63 kb) was amplified with the primer

pair 5′-GCCAGCGTATCGTGCTGCGTTTCGATGCGG-3′ and

5′-CCAGTTGCAACCACCTGTTGATCCGC-3′. Hybridization was

carried out in dextransulphate buffer as described by Sauter

(1997). Membranes were pre-hybridized at 65 °C for 2–3 h,

radioactively labelled probe [(1.0–1.5) × 106 counts per minute

and millilitre of hybridization buffer] was added and hybrid-

ization was continued overnight. Membranes were washed

successively at 65 °C for 10 min in SSC (0.15 M sodium chloride,

0.015 M sodium citrate), and twice for 5 min in 0.5 × SSC

with 0.1% sodium dodecylsulphate (SDS, w/v). Hybridized filters

were exposed to Hyperfilm MP (Amersham Pharmacia Biotech)

at −80 °C for 1–21 days on an eightfold Cronex intensifier

screen (DuPont, Wilmington, DE, USA). For non-radioactive

blots, the digested DNA was transferred on to a Hybond N+

membrane under alkaline conditions as described by the

manufacturer (Amersham). The membrane was hybridized

with an HPT probe labelled by DIG-dUTP using a PCR DIG

(Digoxigenin) Probe Synthesis Kit (Roche Diagnostics,

Mannheim, Germany), the primers 5′-GATCGGACGATT-

GCGTCGCA-3′ and 5′-TATCGGCACTTTGCATCGGC-3′ as

well as plasmid pYF133 (Fang et al., 2002) as template DNA.

Hybridization and DIG detection using CDP-Star were per-

formed as described in the DIG Application Guide for Filter

Hybridization (Roche Diagnostics). The signals were visualized

using Chemiluminescent Detection Film (Roche Diagnostics).

Acknowledgements

We thank P. Eckes (Bayer Crop Science, Frankfurt, Germany)

for providing the p35SPAT plasmid, G. Vancanneyt (Bayer

Crop Science, Gent, Belgium) for the vector p35SGUSINT

and Japan Tobacco Inc. (Higashibara, Japan) for providing

the plasmids pSB1 and pSB11. We are grateful to A. Bam-

mann, B. Betty, M. Gohra, M. Nissen, C. Marthe and I. Otto

for excellent technical assistance. J.K. and L.S. were sup-

ported by the German Bundesministerium für Bildung und

Forschung (grant 0312281A), which we gratefully acknowl-

edge. We are indebted to T. Sharbel for critical reading of

the manuscript.

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